Nehlsen et al: Replicating minicircles
Figure 5. Persistence and expression of plasmid vectors and minicircles in two other cell lines. A. Analyses corresponding to Figure 4 but for HEK293 cells. For the left-hand transfection experiments minicircles and maxicircles were analyzed after 32 and 60 PDs. After 60 PDs cells were subjected to treatments with either 165 nM (R)-Trichostatin A (“TSA”) or 24 mM 5-Aza-cytidine (“AzaC”) as indicated and re-analyzed after an additional 48 h in the presence of these drugs. B. NIH3T3 cells: FACS-analyses for miniand maxicircles after 32 and 55 PDs. For the minicircle the profiles remain nearly unchanged in this interval. For the maxicircle a complete shutoff is noted already after 32 PDs.
have to be overcome if episomal vectors are to be used for the modification of proliferating cells. To be effective it is required that the new genetic material not only replicates but that it is also actively retained through cell division and passed on to daughter cells. These considerations have set the stage for the present study. In past work we have already defined the essential components of an episome that replicates once per cell cycle (Schaarschmidt et al, 2004), i.e. an active transcription unit and a S/MAR while the SV40 origin function was found to be dispensable (Nehlsen 2004). In a computer-assisted way analogous to Figure 2 S/MAR elements were designed such that they can accommodate components of the nuclear scaffold / nuclear matrix, among these scaffold-attachment factor A (SAF-A / hnRNP-U; Jenke et al, 2001, 2004). These interactions mediate the association of the vector with the chromosome arms enabling an effective segregation into the daughter cells (maintenance function, see Bode et al 2001). Other established S/MAR functions are the capacity to reduce epigenetic silencing and to promote histone hyperacetylation (Klehr et al, 1992). Interestingly, the performance of S/MARs can be boosted by the application of histone deacetylase inhibitors such as (R)-Trichostatin A (TSA), butyrate (Schlake et al, 1994) or by certain derivatives (e.g. phenylbutyrate) that have found use for therapeutical applications (Gore et al, 1997). These activities depend – at least in part – on S/MAR
IV. Discussion While there is significant progress in the modification by episomal DNA of slowly-dividing tissues like liver, muscle and brain, maintenance problems have so far limited the use of nonviral episomes for dividing cells, for instance of the hematopoietic system (Papapetrou et al, 2005, 2006). For liver, the most advanced vehicles appear to be “minicircles”, small circular vectors that are exclusively composed from eukaryotic sequences. In contrast to linear DNA, minicircles do not concatemerize and are less prone to integration. It is also known that, owing to their superhelical status, they are better transcriptional templates than linear DNA (Weintraub et al, 1986). Based on this rationale M. A. Kay and coworkers could demonstrate that transgene expression levels in nonreplicating minicircles are not only 45-560 fold higher but also more persistent compared to conventional plasmids (Chen et al, 2003; Riu et al, 2005). The authors applied a critical test to prove the episomal state of these vectors, i.e. a 2/3 hepatectomy upon which almost every hepatocyte undergoes one or two cell doublings until the liver mass is reconstituted. It was shown that during cell cycling the minicircles were lost in accord with their non-integrated (episomal) status (Chen et al, 2001). The results clearly demonstrate that this class of vectors is not functionally attached to chromosomal DNA, which would otherwise provide the required centromere function (Bode et al, 2001) and they anticipate the category of problems that 240